JP2008189622A - SPECIFIC INHIBITOR OF Ca2+/CALMODULIN DEPENDENT PROTEIN KINASE PHOSPHATASE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inhibitor specifically acting on CaMKP and CaMKP-N. <P>SOLUTION: It has been clarified in the invention that Evans Blue, Chicago Sky Blue 6B, Oxamine Blue B and Azo Blue which are color compounds have specific inhibiting action on CaMKP and CaMKP-N, and that 1-amino-8-naphthol-2,4-disulfonic acid and 1-amino-8-naphthol-4-sulfonic acid which are partial constitution units of these color compounds have specific inhibiting action on CaMKP and CaMKP-N. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention, Ca ²⁺ / calmodulin-dependent protein kinase dephosphorylating, protein phosphatase ^{CaMKP (Ca 2+ / calmodulin-dependent} protein kinase phosphatase) and nuclear localized CaMKP of (CaMKP-N) specific Relates to chemical inhibitors.

There are many Ca ²⁺ binding proteins in the cell, and calmodulin plays an important role in the intracellular signal transduction system by Ca ²⁺ among them. Regard regulation of protein phosphorylation in vivo, Ca ²⁺ / calmodulin complex, multifunctional Ca ²⁺ / calmodulin-dependent protein kinase ^{(Ca 2+ / calmodulin-dependent protein} kinase, CaMK) through It plays an important function (Non-Patent Document 1). In particular, Ca ²⁺ / calmodulin-dependent protein kinase II (Ca ²⁺ / calmodulin-dependent protein kinase II, CaMKII) and CaMKIV play important roles in the regulation of higher nervous functions such as memory and the regulation of various cell functions. It has been revealed that The activation mechanism of CaMKII has been shown to be caused by autophosphorylation and activation of Thr286 in the autoinhibition domain, but how it is inactivated after activation. There has been little research on the switch-off mechanism.

Ca ²⁺ / calmodulin-dependent protein kinase phosphatases ^{(Ca 2+ / calmodulin-dependent protein} kinase phosphatase (CaMKP)) is phosphorylated synthetic peptide near autophosphorylation sites Thr286 of CaMKII as good substrates, purified from rat brain Protein phosphatase (Non-patent Document 2). As a result of cDNA sequence analysis, rat-derived CaMKP (rat CaMKP) was a protein having 450 amino acids and a molecular weight of 49165 (GenBank Accession no. AB023634) (Non-patent Documents 3, 4, and 5). CaMKP is a Ser / Thr protein phosphatase that is Mn 2 ⁺ -dependent and insensitive to Okadaic acid / calyculin A, and exists as a monomer unlike PP1 and PP2A. Like PP2A, it is strongly activated by polycations such as polylysine and protamine. CaMKP is a protein phosphatase belonging to the PPM family, but its homology with PP2C alpha is as low as 28%, and there is a large domain not found in PP2C on the N-terminal side. When the tissue distribution of CaMKP was examined, it was found that CaMKP was widely expressed between organs and localized in the cytoplasm. By homology search based on the primary structure of rat CaMKP, this protein was searched in the gene database (GenBank), and it was found that there was a human gene with 82% homology at the gene level and 79% at the amino acid level ( KIAA0015, GenBank Accession No. AF305840) (Non-Patent Document 6). This was a gene with unknown function that had already been obtained by random cloning from human undifferentiated myeloid cell line KG-1, but this was isolated by polymerase chain reaction (PCR) and expressed in E. coli. I let you. As a result of examining its enzymatic properties, it was confirmed that this gene product had the same protein phosphatase activity as rat CaMKP, and it was named human CaMKP (Patent Document 1).

  Based on the primary structure of this human CaMKP, human genes were further searched in the gene database (GenBank), and it was found that there was a human gene that seems to be another homolog (KIAA1072, GenBank accession no. AB028995). . When this gene was acquired and analyzed in detail, this homolog has long regions that are not found in CaMKP at the N-terminus and C-terminus, but there is a region that has 64% homology with human CaMKP centering on the phosphatase domain. Exists (Non-Patent Documents 4, 7, 8). Unlike CaMKP, this human gene was specifically expressed in the brain, and when it was examined in the cell, it was found to be localized in the nucleus. When the enzyme activity was investigated by expressing it in insect cell Sf9, this enzyme showed enzymatic properties similar to CaMKP, such as making phosphorylated CaMKIV a good substrate, so in the sense of nuclear localized CaMKP, Was named CaMKP-N.

CaMKP and CaMKP-N that have been isolated and identified as described above, but the issue of how these roles are divided in vivo and what biological functions are carried out is as follows. It is an important issue. Therefore, we decided to investigate the role of CaMKP and CaMKP-N in the early development of vertebrates by using zebrafish that can be gene knockdown using antisense morpholino oligos as a model animal. First of all, cDNA cloning of zebrafish-derived CaMKP (zCaMKP) was attempted. As a result, zCaMKP was composed of 424 amino acids, and its N-terminal and C-terminal were shorter than rat CaMKP (450 amino acids) and human CaMKP (454 amino acids) (Fig. 1). The enzymatic properties of zCaMKP were optimal for pH 8.0, unlike rat CaMKP, which requires Mn ²⁺ , but rather Mg ²⁺ . zCaMKP lacks an acidic amino acid cluster that is known to be a responsible region for activation by polycations in rat enzymes, and unlike rat CaMKP, it does not undergo activation by polylysine. Rat CaMKP transiently expressed in Neuro2a cells was mostly localized in the cytoplasm, whereas zCaMKP was also present in significant amounts in the nucleus. Thus, although there are some differences compared to rat CaMKP, it was concluded that this enzyme is a zebrafish homologue of CaMKP due to its high substrate specificity to CaMK as with rat CaMKP. In addition, microinjection of antisense morpholino-oligos against zCaMKP into embryos at the 1 to 4 cell stage and the effects on embryo development were examined. Apoptotic cells appeared throughout the body by 72 hours after fertilization, resulting in significant malformations. (Non-Patent Document 5).

  The zebrafish-derived CaMKP-N (zCaMKP-N) cDNA was obtained based on a sequence highly homologous to human-derived CaMKP-N (human CaMKP-N), as in the case of zCaMKP. Compared with the primary structure of human CaMKP-N, the catalytic region was highly conserved, but zCaMKP-N lacked the N-terminal part more than the catalytic region (Fig. 2). In human and rat CaMKP-N, an acidic amino acid cluster, which is responsible for activation by polycations, exists in this region, but in zCaMKP-N, this region moved to the C-terminal side of the catalytic region. In addition, a sequence that seems to be a typical nuclear translocation signal, which is rich in basic amino acids called RKKRRLDVLPLRR, was found in the region of the C-terminal part 575-587 of zCaMKP-N, but this region actually functions as a nuclear translocation signal. (Non-patent document 9). CaMKP-N derived from rat, human and zebrafish acts specifically on CaMK and dephosphorylates in vitro. Among CaMK, CaMKI and CaMKII are mainly localized in the cytoplasm, and CaMKIV is mainly localized in the nucleus. As a result, it was thought that the target of CaMKP-N in the cell might be CaMKIV. Therefore, in order to investigate whether zCaMKP-N dephosphorylates CaMKIV in vivo (intracellular), NeuroCa cells that were expressed with rat CaMKIV alone or co-expressed with zCaMKP-N were stimulated with ionomycin, a calcium ionophore. The phosphorylation levels of CaMKIV were compared between the two. As a result, phosphorylation of Thr196 was remarkably enhanced after stimulation with ionomycin in cells expressing CaMKIV alone, but the phosphorylation level was significantly reduced in cells co-expressed with zCaMKP-N. On the other hand, even when co-expressing mutant D188A having no activity, a decrease in phosphorylation level was not observed (Non-patent Document 9). The above results indicate that zCaMKP-N functions as a negative regulator of CaMKIV not only in vitro but also in vivo.

  Furthermore, gene knockdown with antisense morpholino oligos made the structure of the head unclear, and apoptotic cells stained with acridine orange appeared in the brain, so zCaMKP-N was the first zebrafish in the same way as zCaMKP. It was strongly suggested that it is an enzyme that is essential for development but plays an essential role in the early development of the central nervous system (Non-Patent Document 9).

To analyze the physiological roles of CaMKP and CaMKP-N, the individual level approach (transgenic, knockout mouse, pathological model animal) as described above, the molecular level approach using molecular biology (site-specific) Displacement, etc.) is also important, but in order to elucidate physiological functions using more diverse systems, regulatory molecules that act specifically on these enzymes, especially small molecule inhibitors with cell membrane permeability, are indispensable It is. However, only compounds having an inhibitory effect on PP2C alpha belonging to the same PPM family as CaMKP and CaMKP-N have been reported so far (Non-patent Document 10). Since CaMKP and CaMKP-N are closely related to the regulation of CaMK activity, such specific inhibitors may provide an effective means for the treatment of various diseases and dysfunctions resulting from the failure of CaMK regulation. There is sex.
JP2001-333776 Soderling, T. and Stull, J. (2001) Structure and regulation of calcium / calmodulin-dependent protein kinases. Chem. Rev. 101, 2341-2352 Ishida, A., Kameshita, I. and Fujisawa, H. (1998) A novel protein phosphatase that dephosphorylates and regulates Ca2 + / calmodulin-dependent protein kinase II. J. Biol. Chem., 273, 1904-1910. Kitani, T., Ishida, A., Okuno, S., Takeuchi, M., Kameshita, I. and Fujisawa, H. (1999) Molecular cloning of Ca2 + / Calmodulin-dependent protein kinase phosphatase. J. Biochem., 125 , 1022-1028. Ishida, A., Shigeri, Y., Taniguchi, T., and Kameshita, I. (2003) Protein phosphatases that regulate multifunctional Ca2 + / calmodulin-dependent protein kinases: From biochemistry to pharmacology. Pharmacol. Ther. 100, 291-305 . Noriyuki Sueyoshi, Yasuhiko Ishida, Isamu Kameshita (2005) Protein phosphatase regulating multifunctional Ca2 + / calmodulin-dependent protein kinase Biochemistry 77, 1317-1326. Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K. And Tabata, S. 1994) Prediction of the coding sequences of unidentified human genes.I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1.DNA Res. 1, 27 -35. Takeuchi, M., Ishida, A., Kameshita, I., Kitani, T., Okuno, S. and Fujisawa, H. (2001) Identification and characterization of CaMKP-N, nuclear calmodulin-dependent protein kinase phosphatase. Biochem. 130, 833-840. Tada, Y., Nimura, T., Sueyoshi, N., Ishida, A., Shigeri, Y. and Kameshita, I. (2006) Mutational analysis of Ca2 + / calmodulin-dependent protein kinase phosphatase. Arch. Biochem. Biophys. 452, 174-185. Nimura, T., Sueyoshi, N., Ishida, A., Yoshimura, Y., Ito, M., Tokumitsu, H., Shigeri, Y., Nozaki, N. and Kameshita, I. (2007) Knockdown of nuclear Ca2 + / Calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish. Arch. Biochem. Biophys. 457, 205-216. Rogers, JP, Beuscher, AE IV, Flajolet, M., McAvoy, T., Nairn, AC, Olson, AJ, and Greengard, P. (2006) Discovery of protein phosphatase 2C inhibitors by virtual screening. J. Med. Chem 49, 1658-1667.

  Accordingly, an object of the present invention is to provide an inhibitor that specifically acts on CaMKP and CaMKP-N.

  The present inventor screened compounds that specifically inhibit the function of CaMKP from the compound library, and as a result, two dye compounds, Evans Blue and Chicago Sky Blue 6B, are CaMKP and CaMKP- It has been found that it specifically inhibits N. Further, the present inventor has found that oxamine blue B (Oxamine Blue B) and azo blue (Azo Blue) also specifically show inhibitory activity against CaMKP and CaMKP-N. In addition, the present inventor also has 1-amino-8-naphthol-2,4-disulfonic acid and 1-amino- which are partial structural units of these dyes. It was found that 1-amino-8-naphthol-4-sulfonic acid specifically inhibits CaMKP and CaMKP-N.

  The Evans blue is a compound represented by the following formula (1).

  The Chicago Sky Blue 6B represents a compound represented by the following formula (2).

  The oxamine blue B is a compound represented by the following formula (3).

  The azo blue represents a compound represented by the following formula (4).

  The 1-amino-8-naphthol-2,4-disulfonic acid represents a compound represented by the following formula (5).

  The 1-amino-8-naphthol-4-sulfonic acid represents a compound represented by the following formula (6).

The present invention relates to an inhibitor and a therapeutic agent as follows.
Item 1. A Ca ²⁺ / calmodulin-dependent protein kinase (Ca ²⁺ / calmodulin-dependent protein kinase) containing at least one selected from the group consisting of compounds represented by formulas (1) to (6) inhibitors of phosphatase (CaMKP)) and / or nuclear localized CaMKP (CaMKP-N).
Item 2. A disease caused by inactivation of CaMK by CaMKP and / or CaMKP-N, comprising as an active ingredient at least one selected from the group consisting of compounds represented by formula (1) to formula (6) Therapeutic agent.

  In order to clarify the compounds that specifically inhibit the functions of CaMKP and CaMKP-N, the present inventor examined the inhibitory activity of various compounds. Among many compounds, the compound represented by the formula (1) It was clarified that Evans Blue and Chicago Sky Blue 6B which is a compound represented by the formula (2) specifically inhibit the function of CaMKP. In addition, the inventor of the present invention, Evans Blue, Chicago Sky Blue 6B is a compound similar to Oxamine Blue B which is a compound represented by Formula (3) and Azo Blue which is a compound represented by Formula (4) are also CaMKP. It was found that the function was specifically inhibited. Furthermore, these four dye compounds have been shown to exhibit inhibitory activity against CaMKP-N, a PPM family that has high homology with CaMKP and is localized in the nucleus. At the same time, the present inventor confirmed that these four compounds do not inhibit the protein phosphatase and calcineurin belonging to the PPP family, which is an evolutionarily different family from CaMKP, and act on PP2C alpha belonging to the same PPM family as CaMKP. It was also clarified not to. The experimental results of Example 5 also indicate that these compounds have good cell membrane permeability. Therefore, these four compounds not only act on PPM family protein phosphatases that have not been reported so far, but also act specifically on CaMKP / CaMKP-N in the PPM family, and also on the cell membrane. It is expected to be very promising as an inhibitor having permeability.

  The structures of these four compounds, Evans Blue, Chicago Sky Blue 6B, Oxamine Blue B, and Azo Blue, are roughly divided into two partial constituent units: a central unit and left and right symmetrical units. Therefore, in order to clarify the partial structural units necessary for the inhibitory activity of these dye compounds on CaMKP, 1-amino-8-naphthol-2,4-disulfonic acid (1- amino-8-naphthol-2,4-disulfonic acid, a compound represented by the above formula (5)), 1-amino-8-naphthol-4-sulfonic acid (1-amino-8-naphthol-4-sulfonic acid) acid, a compound represented by the above formula (6)), 1-naphthol-5-sulfonic acid, 4-amino-1-naphthalenesulfonic acid (4-amino-1- naphthalene sulfonic acid), 5-amino-1-naphthol, and 3,3'-dihydroxybenzidine, 3,3'- The activities of CaMKP, CaMKP-N and PP2C alpha were measured using dimethoxybenzidine (3,3'-dimethoxybenzidine) and 3,3'-dimethylbenzidine dihydrochloride. result Only 1-amino-8-naphthol-2,4-disulfonic acid and 1-amino-8-naphthol-4-sulfonic acid showed inhibitory activity against CaMKP, and these two partial units are CaMKP. They also showed inhibitory activity against -N, but they did not show PP2C alpha inhibitory activity for Evans Blue, and compounds similar to the structure at both ends of Evans Blue and those similar to the central structure. The structure is shown in FIG.

  Thus, in the present invention, the site responsible for the specific inhibitory activity of Evans Blue, Chicago Sky Blue 6B, Oxamine Blue B and Azo Blue for CaMKP and CaMKP-N is similar to the partial structural unit of the compound group and the unit. It was also clarified that among the compounds having the structure: 1-amino-8-naphthol-2,4-disulfonic acid and 1-amino-8-naphthol-4-sulfonic acid. In particular, these findings are expected to provide useful information in designing more effective inhibitors with further enhanced inhibition specificity, membrane permeability, and inhibition activity.

  Several effective specific inhibitors, including okadaic acid, have been reported for the PPP family of protein phosphatases, making them an indispensable tool for research into the physiological functions of the PPP family of protein phosphatases. However, with respect to the protein phosphatase of the PPM family, an inhibitor of PP2C alpha has been reported in Non-Patent Document 10 mentioned above. For example, as a compound exhibiting inhibitory activity on PP2C alpha, a metal ion essential for activity is chelated. Except for various chelators, vanadic acid, which has a broad inhibitory activity on phosphatases, has been reported, but regarding CaMKP, compounds such as heparin and NaF that also have a broad inhibitory activity on phosphatases have been reported. Not as many have been reported as inhibitors to the protein phosphatases of the PPP family. This also contributed to the fact that research on the physiological functions of PPM family phosphatases was delayed compared to the PPP family.

  The series of compounds and partial structural units found in this study have no effect on PP2C alpha, which has been considered to be relatively similar at the primary structure level, even in the same PPM family. Therefore, it is considered that these compounds discriminate subtle structural differences between the active centers of CaMKP / CaMKP-N and PP2C alpha. Therefore, the compounds having the above inhibitory activity are not only specific for PPM family phosphatase but also specific for CaMKP and CaMKP-N in the PPM family. However, it is expected to be useful for elucidating physiological functions of CaMKP / CaMKP-N and PPM family phosphatases, whose research has been delayed. Therefore, the present invention proposes an inhibitor of CaMKP and CaMKP-N containing the compound as one of the uses of the compound that specifically inhibits the functions of CaMKP and CaMKP-N.

  CaMKs such as CaMKII and CaMKIV are known to be deeply involved in the control of higher nervous functions such as memory and the onset mechanism of various diseases. CaMKP and CaMKP-N act on CaMK and dephosphorylate them to inactivate them. Therefore, according to the compound that can specifically inhibit the functions of CaMKP and CaMKP-N, it is possible to prevent inactivation of CaMK and to treat or improve various diseases and disorders caused by the failure of CaMK control. it is conceivable that. In fact, Genoux et al. Reported that when overexpressing the inhibitory protein I-1 specific for PP1, a protein phosphatase that regulates the phosphorylation state of CaMKII, using genetic engineering techniques, especially in aging mice Reported that the memory learning effect was significantly improved (Genoux, D., Haditsch, U., Knobloch, M., Michalon, A., Storm, D. and Mansuy, I. (2002) Protein phosphatase 1 is a molecular constraint on learning and memory. Nature, 418,970-975).

  However, since I-1 is a protein, it is difficult to use as a drug, and low molecular weight PP1 inhibitors such as caliculin A usually have strong carcinogenicity, so that it is not practical to use as a medicine. Since CaMKP and CaMKP-N are also important protein phosphatases that control the phosphorylation state of CaMKII, it is expected that specific inhibitors for these may have the same efficacy as PP1 inhibitors. Therefore, the present invention is one of the uses of a compound that specifically inhibits the functions of CaMKP and CaMKP-N. As a use of CaMKP and CaMKP-N, a disease caused by inactivation of CaMK via CaMKP and CaMKP-N is effective. A therapeutic agent is proposed.

  As diseases caused by inactivation of CaMK, diseases (including disorders) that are directly or indirectly caused by inactivation of CaMK, including those that can be known in the future, can be broadly cited. Specific examples include memory impairment (including dementia) and the like due to lowering of higher-order neurological functions and disorders associated with aging as described above, but are not limited thereto.

  Such an inhibitor and therapeutic agent of the present invention may contain any compound that specifically inhibits CaMKP and CaMKP-N described above, and other acceptable carriers, buffers, stabilizers, It does not restrict | limit in particular about mix | blending various additives, such as a coloring agent, a preservative, a fragrance | flavor, a flavor agent, or a sweetener. In addition, the types and amounts of these carriers and additives are not particularly limited as long as they do not interfere with the inhibitory activity of compounds that specifically inhibit CaMKP and CaMKP-N. It is appropriately selected and adopted according to a conventional method.

  Furthermore, the inhibitor of the present invention can be prepared in any form such as solid, semi-solid or liquid. For example, solid inhibitors include tablets, capsules, pills, powders (powder) or granules, and liquid inhibitors include solutions, suspensions or emulsions.

  In addition, the therapeutic agent of the present invention can be designed by appropriately adjusting the administration time, administration route, administration form, dosage and the like according to the type and degree of the target disease, and is not particularly limited thereto. For example, examples of the dosage form include oral administration, parenteral administration, local administration, systemic administration and the like, and according to such administration form, the therapeutic agent of the present invention is in any form such as solid, semi-solid or liquid. Can be prepared. Examples of solid preparations include tablets, capsules, pills, powders (powder) or granules, and liquid preparations include oral solutions, suspensions, emulsions, injections and infusions. Agents (including suspensions and emulsions).

  Hereinafter, the present invention will be described in detail based on examples.

  In the present invention, protein phosphatase activity was measured by the following assay method.

(1) CaMKP activity measurement
Activity was measured using a phosphorylated peptide pp10 (YGGMHRQET (p) VDC) consisting of 12 amino acids imitating the amino acid sequence surrounding the autophosphorylation site of CaMKII as a substrate of rat CaMKP. The reaction solution (50 μl) consists of 50 mM Tris-HCl (pH 8.0), 2 mM MnCl ₂ , 0.1 mM EGTA, 0.01% Tween 20, 20 μM pp10 and CaMKP. The reaction was started by adding rat CaMKP, and the reaction was carried out at 30 ° C. for 6 minutes. Then, malachite green reaction stop solution was added, and free phosphate in the reaction solution was measured by malachite green assay (Baykov, A., Evtushenko, O. and Avaeva, S. (1988) A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171, 266-270).

(2) PP2C alpha activity measurement
Rat PP2C alpha activity was measured by simulating rat CaMKP conditions. Instead of 2 mM MnCl ₂ , 5 mM MgCl ₂ was used.

(3) CaMKP-N activity measurement
The activity of zCaMKP-N was measured by imitating the conditions of rat CaMKP. 40 μM pp10 was used instead of 20 μM pp10.

  In addition, a compound library of Tocris UK (http://www.tocris.com/) was purchased from Tocris.

In the present invention, compounds that specifically inhibit CaMKP and CaMKP-N were determined as follows.
Example 1: Screening from a compound library of Tocris, UK A total of 817 kinds of Tocris compound libraries were added to the enzyme reaction solution so that the final concentration would be 10 μM to 40 μM, respectively, to rat CaMKP and rat PP2C alpha The influence of was investigated. As a result, two types of compounds that specifically inhibit rat CaMKP were found. The two compounds were Evans Blue and Chicago Sky Blue 6B. IC ₅₀ values for rat CaMKP were 4.1 ± 0.2 μM for Evans Blue and 4.1 ± 0.3 μM for Chicago Sky Blue 6B, showing almost the same inhibitory activity. Neither compound showed inhibitory activity on rat PP2C alpha (Table 1).

Example 2 Inhibitory Activity of Evans Blue Analogues To examine whether Evans Blue or Chicago Sky Blue 6B analogues also have inhibitory activity, rat CaMKP and rat PP2C using oxamine blue B and azo blue The activity of alpha was measured. As a result, these two compounds had inhibitory activity against rat CaMKP, but rat PP2C alpha hardly inhibited. IC ₅₀ values for rat CaMKP were 7.9 ± 0.3 μM for oxamine blue and 16.1 ± 1.2 μM for azo blue (Table 1).
Example 3 Inhibitory Activity of Evans Blue Partial Units The structures of Evans Blue, Chicago Sky Blue 6B, Oxamine Blue B, and Azo Blue dye compounds can be broadly divided into a central unit and two symmetrical units. Formed from two partial units. We decided to clarify the partial structural units necessary for the inhibitory activity of these dye compounds on CaMKP. Therefore, the activity of rat CaMKP and rat PP2C alpha was measured using 1-amino-8-naphthol-2,4-disulfonic acid and 1-amino-8-naphthol-4-sulfonic acid, which are similar in structure to both ends of Evans Blue. Went. As a result, both of these two compounds showed inhibitory activity against rat CaMKP. The IC ₅₀ values for these rat CaMKPs were 5.2 ± 0.8 μM for 1-amino-8-naphthol-2,4-disulfonic acid and 3.3 ± 0.2 μM for 1-amino-8-naphthol-4-sulfonic acid. . On the other hand, no compound showed inhibitory activity against rat PP2C alpha.
Example 4: Inhibitory activity against zCaMKP-N
Using zCaMKP-N having high homology to rat CaMKP, it was examined whether the above compounds show inhibitory activity. As a result, it showed inhibitory activity as in the case of rat CaMKP (Table 1). Furthermore, Evans Blue, Chicago Sky Blue 6B, Oxamine Blue B and Azo Blue inhibited zCaMKP-N more strongly than rat CaMKP. IC ₅₀ values for zCaMKP-N of each compound are 0.9 ± 0.1 μM for Evans Blue, 1.0 ± 0.1 μM for Chicago Sky Blue 6B, 2.2 ± 0.3 μM for Oxamine Blue B, 3.0 ± 0.3 μM for Azo Blue, 1-amino- The amount of 8-naphthol-2,4-disulfonic acid was 6.6 ± 0.8 μM, and that of 1-amino-8-naphthol-4-sulfonic acid was 3.8 ± 0.8 μM.
Comparative Example 1-Naphthol-5-sulfonic acid, 4-amino-1-naphthalenesulfonic acid, 5-amino-1-naphthol similar to the structure at both ends of Evans Blue, 3,3'- similar to the central structure The activity of rat CaMKP and rat PP2C alpha was measured using dihydroxybenzidine, 3,3′-dimethoxybenzidine, and 3,3′-dimethylbenzidine dihydrochloride. As a result, none of these compounds showed inhibitory activity against rat CaMKP and rat PP2C alpha.

Furthermore, in the present invention, the inhibitory activity against CaMKP-N expressed in cells was evaluated by the following method. First, 2 × 10 ⁴ mouse neuroblastoma cells Neuro2a were seeded in a 12-well plate and cultured in DMEM containing 10% calf serum for 24 hours. Next, rat CaMKIV expression vector (0.83 μg) and empty vector (pcDNA3.1 (+) / myc-His B, 0.33 μg), or rat CaMKIV expression vector (0.83 μg) and zCaMKP-N expression vector ( 0.33 μg) was introduced into the cells using Lipofectamine 2000. After 24 hours, the cells were treated with the inhibitor Evans Blue or Chicago Sky Blue 6B (100 μM each) dissolved in serum-free DMEM to bring the above-described inhibitors into the cells at the same time as serum starvation. After 6 hours, the medium was further replaced with 1 μM of ionomycin and cultured for 10 minutes. The medium was removed, the reaction was stopped with SDS sample buffer, and after protein quantification, it was subjected to SDS-PAGE. Phosphorylated rat CaMKIV was detected by Western blotting using an anti-phosphorylated rat CaMKIV antibody (α-P-rat CaMKIV).

Example 5
When rat CaMKIV is expressed in Neuro2a cells and Ionomycin is allowed to act, the phosphorylation level of rat CaMKIV increases due to an increase in calcium ion concentration, but when zCaMKP-N is co-expressed there, the phosphorylation level of rat CaMKIV is Decreases significantly (Non-patent Document 9). Therefore, we investigated whether Evans Blue and Chicago Sky Blue 6B can effectively act at the cellular level. A decrease in rat CaMKIV phosphorylation was confirmed in cells co-expressed with zCaMKP-N compared to cells stimulated with rat CaMKIV alone and stimulated with Ionomycin (Fig. 4, comparison between lanes 2 and 5). On the other hand, Evans Blue (Fig. 4, lane 6) or Chicago Sky Blue 6B (Fig. 4, lane 7) was incorporated into cells co-expressed with rat CaMKIV and zCaMKP-N, and these inhibitors were added when stimulated with Ionomycin. The phosphorylation level increased compared to when not (FIG. 4, lane 5). In addition, these inhibitors did not affect the phosphorylation level of CaMKIV when rat CaMKIV was expressed alone and stimulated with Ionomycin (FIG. 4, lanes 2-4). From these results, it was shown that both Evans Blue and Chicago Sky Blue 6B can be taken into cells and exert inhibitory activity.

FIG. 1 shows a comparison of the primary structures of rat, human and zebrafish CaMKP and rat PP2C alpha. FIG. 2 shows a comparison of the primary structures of human CaMKP-N (human CaMKP-N) and zebrafish CaMKP-N (zCaMKP-N). FIG. 3 shows the structure of Evans Blue, and a compound similar to the structure of both ends of Evans Blue and a compound similar to the central structure. FIG. 4 shows the inhibitory activity of Evans Blue and Chicago Sky Blue 6B against CaMKP-N.

Claims (2)

Hereinafter, formula (1) to formula is selected from the group consisting of compounds represented by (6) at least one, Ca ²⁺ / calmodulin-dependent protein kinase phosphatases ^{(Ca 2+ / calmodulin-dependent protein} kinase phosphatase (CaMKP)) and / or an inhibitor of nuclear localized CaMKP (CaMKP-N),

. Hereinafter, a disease caused by inactivation of CaMK by CaMKP and / or CaMKP-N, comprising as an active ingredient at least one selected from the group consisting of compounds represented by formula (1) to formula (6) Therapeutic agent,

.

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